1. Field of the Invention
The present invention relates to a genetically modified microorganism of the genus Corynebacterium producing N-acetal glucosamine, and a method for producing N-acetyl glucosamine or glucosamine using the same.
2. Description of the Related Art
Glucosamine is an amino derivative of glucose, and N-acetyl glucosamine is an acetylated derivative of glucosamine. They are important constituents of many natural polysaccharides, and can form structural materials for cells, constituting the cell wall.
N-acetyl glucosamine is an important component of protein synthesis, involved in tissue regeneration, and thus N-acetyl glucosamine has therapeutic potential in the prevention and treatment of a wide variety of diseases such as gastritis, food allergies, inflammatory bowel disease (IBD), diverticulitis, acute and chronic forms of rheumatoid arthritis and osteoarthritis, as well as the pathological conditions arising from metabolic disorders of the osteoarticular tissues. Glucosamine is also used as a functional food applied for the prevention, and treatment of human, osteoarthritic diseases.
Glucosamine is obtained by acid hydrolysis of chitin, a complex carbohydrate derived from N-acetyl glucosamine. Alternatively, glucosamine can also be produced by acid hydrolysis of chitosans. The raw material, chitin is a copolymer of N-acetyl glucosamine and glucosamine, and is a common natural substance found in arthropods and fungi. The chitin can be obtained from arthropod refuse (lobster, shrimp, krill, crab, and prawn exoskeletons) and more recently from inexpensive sources like fungal biomass used in citric acid production.
Common industrial practice is to purify the chitin by treating it with combinations of acids and bases to remove other impurities accompanying minerals, proteins, etc., and then to depolymerize and deacetylate the chitin in a single step into glucosamine through the use of concentrated hydrochloric acid in low yields at high temperature for a long time. Glucosamine as a free base is very unstable and subject to degradation. Consequently, stable salts such as hydrochloride are produced.
The glucosamine content in arthropod refuse and fungal biomass is low and thus large volumes of waste are produced. Glucosamine is considerably expensive because the production process itself has relatively low yield and is energy and chemically intensive. In addition, with the common forms of glucosamine, being derived from shellfish, it is known that there is the potential for allergic reactions in persons sensitive to shellfish. Moreover, the availability of raw material (e.g., a source of chitin, such as crab shells) becomes gradually limited. Therefore, there is a need for a cost-effective method for producing glucosamine and N-acetyl glucosamine in high yields for commercial sale and use.
PCT Publication No. WO 02/66667 discloses a method of making glucosamine from microbial biomass to remove the possibility of allergic reactions. This production method overcomes problems associated with shellfish allergy, but it suffers from a major problem of low yield. More particularly, since the method relies on the biomass waste generated in fermentation of other products such as citric acid, it is not enough to produce sufficient quantities of glucosamine that meet the increasing market demand for the product.
Meanwhile, N-acetyl glucosamine is currently produced by the acetylation of glucosamine using an organic acetylating reagent such as acetic anhydride, but this production method requires high costs. Also, because of low production yield, it is not easy to produce N-acetyl glucosamine on a large scale.
To overcome these problems, U.S. Pat. No. 7,332,304 discloses a method of producing N-acetyl glucosamine by E. coli fermentation. This method suggests E. coli fermentation for a high production yield of N-acetyl glucosamine which dramatically resolves the encountered problems including low production yield of N-acetyl glucosamine due to the conventional complicated process. In addition, it is known that glucosamine may be produced from deacetylation of N-acetyl glucosamine by simple acid treatment (Novikov V. Y. et al. Russ. J. Appl. Chem. 1997:1467-1470), and thus it is also possible to produce glucosamine in a high yield.
However, this method is not suitable for the production of health foods, because the use of E. coli generates potentially toxic endotoxins. In addition, glucosamine-6-phosphate acetyltransferase, essential for the production of N-acetyl glucosamine (see FIG. 1: GNA1), does not exist in E. coli, and thus other microorganisms such as yeast should be introduced. There is a limitation in that expression of the crucial enzyme requires an inducible expression system (Deng M. D. et al. Met Eng. 2005:201-214). Unregulated expression of the crucial enzyme causes severe inhibition of cell proliferation. The inducible expression system is disadvantageous in that an inducing material is needed, leading to an increase in production costs and an induction time point should be determined, and therefore it is considered to be not suitable for industrial production by large-scale fermentation. In terms of safety of production and possibility of industrial adaptation, the above method has a drawback of using E. coli and an inducible expression system.
Until now, E. coli fermentation technology has been developed in order to overcome the problems of the limited availability of raw materials and low production yield. However, there have been no strains, suitable for producing N-acetyl glucosamine and glucosamine, which are safe for humans and animals. Further, there have been no reports of a producing strain that utilizes a constitutive expression system other than an inducible expression system to facilitate industrial enzyme production.
In order to solve the above problems, the present inventors have developed a microorganism of the genus Corynebacterium for large-scale fermentation of N-acetyl glucosamine and glucosamine without causing environmental problems, capable of realizing production stability and industrial application, and a method for producing N-acetyl glucosamine and glucosamine using the same, thereby completing the present invention.